Significant amounts of carbon dioxide are generated by mammals such as humans. In general, only a small portion of the CO.sub.2 generated is utilized; the rest is considered waste material and must be eliminated. One means for such elimination is through body fluids, such as the blood stream. The concentration of carbon dioxide in the blood stream has a profound effect on body functions. Thus, the measurement of carbon dioxide content in the blood stream or other body fluids is an important tool for precise and accurate medical diagnosis.
The term carbon dioxide content, as applied to body fluids means the sum of bicarbonate ions, carbonic acid and dissolved carbon dioxide according to the equilibrium EQU CO.sub.2 +H.sub.2 O.revreaction.H.sub.2 CO.sub.3 .revreaction.H.sup.+ +HCO.sub.3.sup.-
U.S. Pat. No. 3,974,037 discloses several enzymatic methods for the determination of carbon dioxide in body fluids. These include assays involving either reactions (1), (2), (3) or (4) below, each generating oxalacetate which can be measured with reaction (5) as follows: ##STR1## where PEP carboxylase is phosphoenolpyruvate carboxylase and P.sub.i is inorganic phosphorus; ##STR2## where ADP stands for adenosine diphosphate and ATP stands for adenosine triphosphate. In the reaction defined by (3), the ADP can be replaced by other nucleotide diphosphates depending on the source used to prepare the PEP carboxykinase.
An additional possible enzymatic assay for carbon dioxide is the following reaction involving phosphoenolpyruvate carboxykinase(pyrophosphate) and inorganic phosphorus: ##STR3## where PP.sub.i stands for inorganic pyrophosphate; and ##STR4## where MDH is malate dehydrogenase, NADH is the reduced form of nicotinamide adenine dinucleotide and NAD is the oxidized form.
Each of (1), (2), (3) or (4) when coupled with reaction (5) measures carbon dioxide content either as CO.sub.2 or HCO.sub.3.sup.- by determining the concentration change in NADH while maintaining a constant pH. The assay is carried out by mixing all of the reagents required by (1) and (5), (2) and (5), (3) and (5) or (4) and (5) with measured amounts of the body fluid sample, typically a body fluid such as serum or plasma.
When samples of body fluids contain any blood, hemolysis often occurs; that is, the rupture of erythrocytes or red blood cells. The process of hemolysis, therefore, results in the release of all the contents in the erythrocytes including hemoglobin and carbonic anhydrase into the body fluid. Measurement of hemoglobin is, therefore, a direct measure of the extent of hemolysis and is also an indicator of the relative amount of carbonic anhydrase released into the body fluid. Carbonic anhydrase shifts, to a variable extent, the HCO.sub.3.sup.- to CO.sub.2 equilibrium in body fluids since it catalyzes the following reaction EQU H.sup.+ +HCO.sub.3.sup.- .revreaction.CO.sub.2 +H.sub.2 O.
Enzymatic measurements for total carbon dioxide using hemolyzed samples using reactions (1)-(4) results in a bias in the assay for the total CO.sub.2 as compared to non-hemolysed. The bias is a result of a shift in the equilibrium brought about by carbonic anhydrase. The bias can be negative or positive depending on selected reactions (1)-(4). Normal serum or plasma also contain low levels of circulating carbonic anhydrase which contributes to the random bias seen with existing enzymatic CO.sub.2 procedures.
U.S. Pat. No. 3,974,037 teaches that the accuracy of the enzymatic assay procedures disclosed therein can be increased by the addition of carbonic anhydrase to test solutions.
Contrariwise, our research has shown that the presence of carbonic anhydrase, endogenous or added, in body fluids causes unacceptable inaccuracies in an enzymatic assay for total CO.sub.2.